Method for selecting 1-deoxy-D-xylulose-5-phosphate synthase inhibitor from plant extract

A plant extract and inhibitor technology, applied in the field of enzyme inhibitor screening, can solve problems such as large-scale and expensive, and achieve the effects of strong feasibility, high sensitivity and good safety

Inactive Publication Date: 2013-01-30
NORTHWEST UNIV(CN)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The object of the present invention is to provide a simple and fast method for screening DXS enzyme inhibitors from plant extracts, which is suitable for extracting Inhibitors of DXS enzymes are screened in substances, which solves the technical problems of screening DXS enzyme inhibitors in the background technology that require large and expensive instruments, such as nuclear magnetic resonance instruments, etc., or require radioactive detection.

Method used

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  • Method for selecting 1-deoxy-D-xylulose-5-phosphate synthase inhibitor from plant extract
  • Method for selecting 1-deoxy-D-xylulose-5-phosphate synthase inhibitor from plant extract
  • Method for selecting 1-deoxy-D-xylulose-5-phosphate synthase inhibitor from plant extract

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preparation example Construction

[0038] 2) Preparation of control samples

[0039] 2-10mM D-GAP (or DHAP), 5-20mM Mg 2+ or Zn 2+ , 0.2-2mg / ml of DXS enzyme is added to 40-400mM Tris-HCl or PBS (pH5.0-9.0) buffer solution, and the reaction system can prepare the control sample;

[0040] The difference between the sample prepared in step 1) for detecting DXS enzyme inhibitors and the control sample prepared in step 2) is that no plant extract is added to the control sample, and the other components are the same.

[0041] In actual experimental operation, the total volume of the reaction system in step 1) and step 2) is respectively 50-200 μ l (in the control sample of step 2), no plant extract is added, and the volume is replenished with ultrapure water), the volume of The selection can ensure that the reaction can be completely carried out under the condition of detection, and the substrate will not be wasted. The reaction temperature is 30-40°C, which ensures the activity of the enzyme. The reaction time is...

specific Embodiment 1

[0056] Specific Example 1: Using D-GAP as a substrate

[0057] 1. Isomerization reaction

[0058] 1) Preparation of samples for detection of DXS enzyme inhibitors:

[0059] 5mM D-GAP, 20mM Mg 2+ , 0.5mg / ml of DXS enzyme was added to 60mM Tris-HCl (pH8.0) buffer, and then 20μg / μl of plant extract was added, and the reaction system was reacted in a water bath at 37°C for 2h to prepare a Samples of DXS enzyme inhibitors;

[0060] 2) Preparation of control samples

[0061] A control sample was prepared in the same manner as above, and the reactants and concentrations in the control sample were the same as in 1), the difference being that no plant extract was added to the control sample, and the volume was supplemented with ultrapure water.

[0062] 2. Enzyme digestion reaction

[0063] After the isomerization reaction, take 50 μl of the reaction product in 1, add 6 μl of 10×CIAP buffer, add 1 unit of alkaline phosphatase, and incubate at 37°C for 2 hours.

[0064] 3. Derivat...

specific Embodiment 2

[0072] Specific embodiment two: take DHAP as substrate

[0073] 1. Isomerization reaction

[0074] 1) Preparation of samples for detection of DXS enzyme inhibitors:

[0075] 5mM DHAP, 20mM Mg 2+ , 0.5mg / ml of DXS enzyme was added to 60mM Tris-HCl (pH8.0) buffer, and then 20μg / μl of plant extract was added, and the reaction system was reacted in a water bath at 37°C for 2h to prepare a Samples of DXS enzyme inhibitors;

[0076] 2) Preparation of control samples

[0077] A control sample was prepared in the same manner as above, and the reactants and concentrations in the control sample were the same as in 1), the difference being that no plant extract was added to the control sample, and the volume was supplemented with ultrapure water.

[0078] 2. Enzyme digestion reaction

[0079] After the isomerization reaction, take 50 μl of the reaction product in 1, add 6 μl of 10×CIAP buffer, add 1 unit of alkaline phosphatase, and incubate at 37°C for 2 hours.

[0080] 3. Derivat...

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Abstract

The invention relates to a method for selecting a 1-deoxy-D-xylulose-5-phosphate synthase inhibitor from a plant extract. The inbibitional effect of the added plant extract on phosphotriose isomerization activity of DXS enzyme is assessed by detecting the conversion quantity of a substrate with pre-column derivatization-high performance liquid chromatography is evaluated. The method mainly comprises the following steps of: adding the plant extract in a phosphotriose isomerization reaction system catalyzed by the DXS enzyme; after reaction at a certain temperature, hydrolyzing the phosphotriose in the reaction system with phosphatase; and next, adding 2,4-dinitrophenylhydrazine to the reaction system for derivatization and then performing detection by using an ultraviolet detector and taking methanol-water or acetonitrile-water as eluent gradients with high performance liquid chromatography. In the selection process of the method, the substrate D-glyceraldehyde 3-phosphoric acid (D-GAP) or dihydroxyacetone phosphate(DHAP) contains no isotope labeling; and the selection process is simple, quick and high in feasibility without any expensive large instrument for detection.

Description

technical field [0001] The invention belongs to the field of enzyme inhibitor screening, in particular to a simple method for screening 1-deoxy-D-xylulose 5-phosphate synthase (DXS enzyme) inhibitors from plant extracts. Background technique [0002] Terpenoids, also known as isoprenoids, are a class of natural hydrocarbon compounds that widely exist in living organisms. They play an important role in living organisms, such as phytosterols participate in the construction of biofilms; ubiquinones participate in respiration; The side chains and plastoquinones of carotenoids and chlorophyll participate in photosynthesis; estrogen participates in the process of intercellular signal transmission and can control the growth and development of individuals; dolichol and isoprenyl groups participate in protein modification; capsidiol (capsidiol ) can be used as antibiotics and phytoalexins for mutual defense between species; gibberellins, abscisic acid, cytokinins and brassinolides re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/42G01N30/89G01N30/02
Inventor 高文运李恒李瑾胡玥秦巍
Owner NORTHWEST UNIV(CN)
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